THE ROLE OF EF-2 KINASE IN DRUG RESISTANCE

我们的长期目标是研究蛋白质合成在程序性细胞死亡中的作用。已知蛋白质合成的抑制可阻断程序性细胞死亡并保护肿瘤细胞免受许多抗肿瘤药物的细胞毒性作用。在当前的提案中，我们计划研究eEf-2激酶在调节蛋白质合成和程序性细胞死亡中的作用。eEF-2激酶，一种普遍存在的蛋白激酶，其功能是磷酸化延伸因子-2（eEF-2）并阻止蛋白合成（Ryazanov等（1988）Nature 334：170-173）。我们最近对来自不同物种的eEF-2激酶进行了克隆和测序，发现它是一类新型蛋白激酶的成员，这些蛋白激酶在结构上和进化上与常规真核蛋白激酶无关（Ryazanov et.等人（1997）Proc. Sci. USA 94：4884-4889）。在初步研究中，我们已经获得证据表明，在许多人类肿瘤中发现的eEF-2激酶表达增加可以使细胞对抗癌药物产生耐药性。此外，我们发现eEF-2激酶被pH值的轻微降低强烈激活，这是缺氧实体瘤中常见的一种情况。我们假设，pH依赖性激活的eEF-2激酶，阻止蛋白质的合成，是一个主要的保护机制的细胞对程序性细胞死亡和抗癌药物的细胞毒性作用。因此，预期eEF-2激酶的抑制使细胞对凋亡刺激和抗癌药物敏感。我们的最终目标是开发eEF-2激酶的特异性抑制剂，以使实体瘤对程序性细胞死亡敏感。具体而言，eEF-2激酶在程序性细胞死亡中的作用将通过构建eEF-2激酶敲除小鼠来评估。将分析缺乏eEF-2激酶的细胞对抗癌药物的抗性/敏感性。将进行机制研究以揭示eEF-2激酶在凋亡信号转导级联网络中的确切位置。 我们将研究eEF-2激酶的分子特性，这是一个潜在的药物靶点。在初步研究中，我们已经表明，eEF-2激酶有一个不寻常的催化结构域，并可能识别其底物中的α-螺旋构象。将进行实验以设计和开发可用作促凋亡剂的特异性eEF-2激酶抑制剂。

Our long-term goal is to study the role of protein synthesis in programmed cell death. Inhibition of protein synthesis is known to block programmed cell death and protect tumor cells from the cytotoxic action of many anti-tumor drugs. In the current proposal, we plan to investigate the role of eEf-2 kinase in regulating protein synthesis and programmed cell death. eEF-2 kinase, a ubiquitous protein kinase, whose function is to phosphorylate elongation factor-2 (eEF-2) and arrest protein synthesis (Ryazanov et al. (1988) Nature 334: 170-173). We recently cloned and sequenced eEF-2 kinase from various species and discovered that it is a member of a novel class of protein kinases that are structurally and evolutionarily unrelated to conventional eukaryotic protein kinases (Ryazanov et. al (1997) Proc. Nat. Acad. Sci. USA 94: 4884-4889). In preliminary studies, we have obtained evidence that increased eEF-2 kinase expression, which has been found in many human tumors, can confer resistance of cells to anti-cancer drugs. In addition, we found that eEF-2 kinase is strongly activated by a slight decrease in pH, a condition normally found in hypoxic solid tumors. We hypothesize that pH- dependent activation of eEF-2 kinase, which arrests protein synthesis, is a major protective mechanism of cells against programmed cell death and the cytotoxic action of anti-cancer drugs. Consequently, inhibition of eEF-2 kinase is expected to sensitize cells against apoptotic stimuli and anti-cancer drugs. Our ultimate goal is to develop specific inhibitors of eEF-2 kinase in order to sensitize solid tumors to programmed cell death. Specifically, the role of eEF-2 kinase in programmed cell death will be evaluated by construction of eEF-2 kinase knockout mice. The resistance/sensitivity of cells lacking eEF-2 kinase to anti-cancer drugs will be analyzed. Mechanistic studies will be performed to reveal the exact position of eEF-2 kinase in the network of apoptotic signal transduction cascades. We will investigate the molecular properties of eEF-2 kinase a potential drug target. In preliminary studies, we have shown that eEF-2 kinase has an unusual catalytic domain, and may recognize alpha-helical conformation in its substrate. Experiments will be performed to design and develop specific eEF-2 kinase inhibitors which can serve as pro- apoptotic agents.